The nature of the genes and proteins responsible for the expression of the neoplastic phenotype of chemically transformed human cells has been investigated by using molecular cloning and other new techniques in molecular biology. A new polypeptide recognized in a chemically transformed human cell line has been identified as a mutant of beta-actin. The synthesis of the mutated beta-actin was correlated with the expression of the transformed phenotype in the variants of the transformed line and its hybrids with normal human fibroblasts. A mutation resulted in several defects in the function of beta-actin, such as increased instability, reduced incorporation into cytoskeletal elements, and decreased ability to polymerize in vitro. These defects in the beta-actin molecule were associated with the disruption and loss of the structural organization of the cytoskeleton, such as the actin cable network. The results suggest that a mutation in beta-actin leads the cells to express transformation by disrupting the cytoskeletal structure and its function. Two smooth muscle actin genes, stomach type and aorta type, were first isolated from human fibroblast DNA. They contain DNA sequences which completely match the amino acid sequences of the corresponding actin polypeptides. Comparison of the intron location in all actin genes in all species indicate that both deletion and insertion of the intron have been involved in the evolution of the actin gene family.